Background: Traumatic brain injury (TBI) occurs in individuals of all ages, predominantly during sports, accidents, and in active military service members. Chronic consequences of TBI include declined cognitive and motor function, dementia, and emotional distress. Small extracellular vesicles (sEVs), previously referred to as exosomes, are nano-sized lipid vesicles that play a role in intercellular communication. Our prior research established the efficacy of sEVs derived from human adipose stem cells (hASC sEVs) in accelerating the healing of brain injuries. The hASC sEVs are a biologic therapeutic and need to be stored at −20 °C or −80 °C. This limits their use in translating to everyday use in clinics or their inclusion in first-aid kits for application immediately after injury. To address this, here we demonstrate that hASC sEVs can be stored at room temperature (RT) for two months post lyophilization. Methods: A transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) were used to validate the morphology of lyophilized RT sEVs. Using in vitro models of neuronal injury mimicking physical injury, inflammation, and oxidative stress, we demonstrate that lyophilized RT hASC sEVs are viable and promote the healing of neuronal injuries. Results: The lyophilized sEVs maintain their purity, size, and morphology upon rehydration. Lyophilized, RT stored sEVs showed better efficacy after two months compared with −80 °C stored sEVs. Conclusions: RT storage of lyophilized hASC sEVs maintains their efficacy to accelerate the healing of injuries in neuronal cells. This will advance the use of hASC sEVs, bringing them closer to use in clinics, home first-aid kits, and on battlefields by active service members.

Wound healing progresses through four phases: hemostasis, inflammation, proliferation, and remodeling. Wounds may become chronic if this process is disrupted. The use of small extracellular vesicle (sEV; EVs < 200 nm) exosomes (exo; ~40–120 nm) derived from human adipose stem cells (hASCs) as a treatment for wounds is well studied. The cargo of these exosomes is of great interest as this accelerates wound healing. Our previous studies identified lncRNAs GAS5 and MALAT1 as packaged and enriched in hASC exosomes. In this study, we use a rat model to examine the effects on wound healing when hASC exosomes are depleted of GAS5 and MALAT1. Rats were wounded and wounds were treated with 100 μg hASC_exo or hASC_exo-G-M every 2 days for 1 week. qPCR was completed to evaluate the molecular effects of depletion of GAS5 and MALAT1 from hASC_exo. RNAseq was performed on wound tissue to evaluate the molecular mechanisms changed by hASC_exo-G-M in wound healing. While hASC_exo-G-M significantly improved wound healing rate compared to control wounds, healing occurred slower than in wounds treated with hASC_exo that were not depleted of GAS5 and MALAT1. Overall, this study reveals that molecular functions associated with healing are reduced in the absence of GAS5 and MALAT1, highlighting the importance of these lncRNAs.

Human intraperitoneal omental adipose tissue, part of the visceral adipose depots, surrounds the abdominal organs and has functions distinct from the subcutaneous adipose depots. In the clinical setting, it is observed that the omentum is beneficial to combat internal sources of inflammation, oxidative stress, and injury-related stress. However, the molecular mechanisms involved in these functions are not fully understood. We previously demonstrated that adipose stem cells derived from human omental adipose tissue (om-hASCs) secrete exosomes (exos). We and others have extensively evaluated the subcutaneous adipose depot-derived exosomes; however, the role of adipose stem cells derived from the human omental depot (om-hASCs) remains less known. In this study, we postulated that exosomes from om-hASCs (om-hASCexos) drive the repair ability of the omentum to heal organs after internal injury and insults. First, we characterized the om-hASCexos using a proteomic analysis which identified the distinct cargo. Using in vitro injury models, we show that om-hASCexos significantly improve cell migration and proliferation, while decreasing oxidative stress and inflammation. To study acute in vivo healing, a rat wound model was evaluated. Om-hASCexos significantly improved the healing rate of injuries. RNAseq revealed that om-hASCexo treatment acts upon pathways associated with lipid and fatty acid metabolism, apoptosis, immune response, and cell differentiation. The pathway analysis indicated that om-hASCexos significantly regulate the expression of Clec5a and Trem1 in the immune response pathway. Overall, we demonstrate the singular properties of om-hASCexos that are distinct from other sources of hASC. Thus, this study provides an understanding of the unique ability of the omental adipose depot to combat internal injuries.

Stimulator of Interferon Genes (STING) is a central mediator of innate immune signaling and a therapeutic target for inflammatory diseases. Previously, we identified Clonixeril (CXL) as a concentration-dependent inhibitory modulator of human STING (hSTING) signaling. Despite structural conservation, murine STING (mSTING) and hSTING differ in ligand recognition and activation. We posit that CXL may exhibit distinct activity in the murine system. Mice are used in biomedical research due to their genetic similarities to humans and their utility in evaluating systemic activity and safety of therapeutic compounds. We evaluated CXL effects on mSTING using computational modeling, as well as in vitro and in vivo studies. Molecular dynamics simulations and induced-fit docking predicted favorable CXL binding to mSTING. In 3T3-L1 cells, CXL enhanced DMXAA-induced STING, TBK1, and IRF3 phosphorylation and IFN-β mRNA expression, showing STING pathway activation. Acute CXL efficacy was evaluated in vivo in C57BL/6 mice. CXL increased IFN-β following DMXAA administration. Histopathology showed no treatment-associated abnormalities. HPLC detected CXL in serum and liver. The study identified species-specific responses, suggesting that mouse models may not accurately reflect the effects of CXL on hSTING signaling. Acute CXL treatment was well-tolerated, and CXL is a positive modulator of DMXAA-induced mSTING signaling in vivo.

Dysregulation of target genes of nonsense-mediated decay (NMD) in the brain remains sparsely known in tauopathies. PS19 transgenic mice expressing human mutant P301S tau were evaluated for levels of lncRNA Gas5, a target of NMD. The results show Gas5 decreased in the brains of PS19 mice as they aged. We evaluated the consequences of blocking the NMD-mediated turnover of Gas5 using a small molecule administered intranasally to PS19 mice. The results show NPC86 disassociated Upf1 and Gas5, thereby hindering NMD. NPC86 treatment increased Gas5 levels in the cortex of male PS19 mice concurrent with a highly significant decrease in pTau S214 and neuroinflammatory genes while increasing insulin signaling. Consequently, digital spatial profiling identified Gas5-regulated genes and pathways. NPC86 treatment enhanced neuronal homeostasis, synaptic vesicle transport and mitochondrial function and downregulated neuroinflammatory pathways. The nodal genes in Parkinson’s signaling pathway, the multiple sclerosis signaling pathway, the Gα(s) signaling pathway, the neuroinflammation signaling pathway, and the interferon gamma signaling pathway were decreased in response to NPC86. The study demonstrates the potential of selectively stabilizing NMD-target lncRNA Gas5 levels to alleviate early neurodegenerative pathology in tauopathy.

US Veterans are disproportionately affected by type 2 diabetes (T2D) compared with civilians. Previously, we demonstrated that serum GAS5 levels are lower in Veterans with T2D compared with non-diabetics and established GAS5 as a genetic biomarker for T2D, validated by others. Dried blood spots (DBS) are advantageous because collection is minimally invasive, and shipment can be at room temperature. Measurement of lncRNAs in DBS is not yet reported and hence we sought to measure lncRNA GAS5 levels in DBS collected from Veteran participants. Results demonstrate that older age was independently associated with lower GAS5 levels among Veterans. Comparing Veterans across war eras, in this cohort, Vietnam War (VW) Veterans had higher incidences of diabetes compared to Gulf War (GW) or Other (non-VW/GW) wars. VW Veterans with A1c values greater than 6.5 had lower GAS5 levels, independent of BMI. Among the VW Veterans, a very small number of Veterans with self-reported Agent Orange (AO) exposure had lower GAS5 levels. Based on this observation, a mouse model with acute TCDD exposure was evaluated for Gas5 levels. The results demonstrate a significant negative correlation between TCDD dose and Gas5 levels measured in blood and DBS in the mouse model. Overall, the study demonstrates that lncRNA GAS5 measured in DBS and blood may be used in biomarker studies in diabetic patients. These results support the use of DBS in longitudinal studies of chronic diseases in active-duty service members or Veterans with toxic exposures during military service.